Label-Free Proteomics of a Defined, Binary Co-culture Reveals Diversity of Competitive Responses Between Members of a Model Soil Microbial System.

Interactions among members of microbial consortia drive the complex dynamics in soil, gut, and biotechnology microbiomes. Proteomic analysis of defined co-cultures of well-characterized species provides valuable information about microbial interactions. We used a label-free approach to quantify the responses to co-culture of two model bacterial species relevant to soil and rhizosphere ecology, Bacillus atrophaeus and Pseudomonas putida. Experiments determined the ratio of species in co-culture that would result in the greatest number of high-confidence protein identifications for both species. The 281 and 256 proteins with significant shifts in abundance for B. atrophaeus and P. putida, respectively, indicated responses to co-culture in overall metabolism, cell motility, and response to antagonistic compounds. Proteins associated with a virulent phenotype during surface-associated growth were significantly more abundant for P. putida in co-culture. Co-culture on agar plates triggered a filamentous phenotype in P. putida and avoidance of P. putida by B. atrophaeus colonies, corroborating antagonistic interactions between these species. Additional experiments showing increased relative abundance of P. putida under conditions of iron or zinc limitation and increased relative abundance of B. atrophaeus under magnesium limitation were consistent with patterns of changes in abundance of metal-binding proteins during co-culture. These results provide details on the nature of interactions between two species with antagonistic capabilities. Significant challenges remaining for the development of proteomics as a tool in microbial ecology include accurate quantification of low-abundance peptides, especially from rare species present at low relative abundance in a consortium.

Characterization of the relative importance of human- and infrastructure-associated bacteria in grey water: a case study.

AIMS: Development of efficacious grey water (GW) treatment systems would benefit from detailed knowledge of the bacterial composition of GW. Thus, the aim of this study was to characterize the bacterial composition from (i) various points throughout a GW recycling system that collects shower and sink handwash (SH) water into an equalization tank (ET) prior to treatment and (ii) laundry (LA) water effluent of a commercial-scale washer. METHODS AND RESULTS: Bacterial composition was analysed by high-throughput pyrosequencing of the 16S rRNA gene. LA was dominated by skin-associated bacteria, with Corynebacterium, Staphylococcus, Micrococcus, Propionibacterium and Lactobacillus collectively accounting for nearly 50% of the total sequences. SH contained a more evenly distributed community than LA, with some overlap (e.g. Propionibacterium), but also contained distinct genera common to wastewater infrastructure (e.g. Zoogloea). The ET contained many of these same wastewater infrastructure-associated bacteria, but was dominated by genera adapted for anaerobic conditions. CONCLUSIONS: The data indicate that a relatively consistent set of skin-associated genera are the dominant human-associated bacteria in GW, but infrastructure-associated bacteria from the GW collection system and ET used for transient storage will be the most common bacteria entering GW treatment and reuse systems. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to use high-throughput sequencing to identify the bacterial composition of various GW sources.

Quantification of genes and gene transcripts for microbial perchlorate reduction in fixed-bed bioreactors.

AIMS: Optimization of full-scale, biological perchlorate treatment processes for drinking water would benefit from knowledge of the location and quantity of perchlorate-reducing bacteria (PRB) and expression of perchlorate-related genes in bioreactors. The aim of this study was to quantify perchlorate removal and perchlorate-related genes (pcrA and cld) and their transcripts in bioreactors and to determine whether these genes or transcripts could serve as useful biomarkers for perchlorate treatment processes. METHODS AND RESULTS: Quantitative PCR (qPCR) assays targeting pcrA and cld were applied to two pilot-scale, fixed-bed bioreactors treating perchlorate-contaminated groundwater. pcrA and cld genes per microgram of DNA were two- to threefold higher and three- to fourfold higher, respectively, in the bioreactor showing superior perchlorate-removal performance. In a laboratory-scale bioreactor, quantities of pcrA and cld genes and transcripts were compared under two distinct performance conditions (c.60 and 20% perchlorate removal) for a 5-min empty bed contact time. cld genes per microgram of DNA were approximately threefold higher and cld transcripts per microgram of RNA were approximately sixfold higher under the higher perchlorate-removal condition. No differences in pcrA genes or transcripts per microgram of DNA or RNA, respectively, were detected between the c.60 and 20% perchlorate-removal conditions, possibly because these assays did not accurately quantify pcrA genes and transcripts in the mixed culture present. CONCLUSIONS: Quantities of cld genes and transcripts per microgram of DNA and RNA, respectively, were found to be higher when perchlorate removal was higher. However, quantities of pcrA and cld genes or transcripts were not found to directly correlate with perchlorate-removal rates. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this study represents the first application of qPCR assays to quantify perchlorate-related genes and transcripts in continuous-flow bioreactors. The results indicate that cld gene and transcript quantities can provide insights regarding the quantity, location and gene expression of PRB in bioreactors.

Loss of translational control in yeast compromised for the major mRNA decay pathway.

The cytoplasmic fate of mRNAs is dictated by the relative activities of the intimately connected mRNA decay and translation initiation pathways. In this study, we have found that yeast strains compromised for stages downstream of deadenylation in the major mRNA decay pathway are incapable of inhibiting global translation initiation in response to stress. In the past, the paradigm of the eIF2alpha kinase-dependent amino acid starvation pathway in yeast has been used to evaluate this highly conserved stress response in all eukaryotic cells. Using a similar approach we have found that even though the mRNA decay mutants maintain high levels of general translation, they exhibit many of the hallmarks of amino acid starvation, including increased eIF2alpha phosphorylation and activated GCN4 mRNA translation. Therefore, these mutants appear translationally oblivious to decreased ternary complex abundance, and we propose that this is due to higher rates of mRNA recruitment to the 40S ribosomal subunit.

A novel eIF2B-dependent mechanism of translational control in yeast as a response to fusel alcohols.

Fusel alcohols are natural products of amino acid catabolism in the yeast Saccharomyces cerevisiae that cause morphological changes similar to those seen during pseudohyphal growth. We have discovered that certain of these alcohols, including butanol and isoamyl alcohol, bring about a rapid inhibition of translation at the initiation step. This inhibition is strain specific and is not explained by previously described translational control pathways. Using genetic mapping, we have identified a proline to serine allelic variation at amino acid 180 of the GCD1 gene product as the genetic locus that allows translational regulation upon butanol addition. Gcd1p forms part of the eIF2B guanine nucleotide complex that is responsible for recycling eIF2-GDP to eIF2-GTP. This represents one of the key limiting steps of translation initiation and we provide evidence that fusel alcohols target eIF2B in order to bring about translational regulation.

New 1-aryl-4-(biarylmethylene)piperazines as potential atypical antipsychotics sharing dopamine D(2)-receptor and serotonin 5-HT(1A)-receptor affinities.

This paper describes the syntheses of several 1-aryl-4-(biarylmethylene)piperazines and the results of the determination of their affinity for D(2) and 5-HT(1A) receptors. A selection of these compounds was evaluated in vivo, resulting in the identification of a drug candidate which is being clinically evaluated as a potential atypical antipsychotic with reduced extrapyrimidal side effects.

Commercial health plan participation in Medicaid managed care: an examination of six markets.

This study examines six local health care markets to gain a better understanding of the factors associated with the decision by commercial plans to participate in Medicaid managed care (MMC). Findings suggest that no single factor explained why plans chose to participate in MMC in a particular market. Instead, a combination of factors--generally economic but not always--determined whether a plan participated. While rate adequacy was central, it was not the only factor. Results indicate that it is capitation rates relative to other factors (such as provider costs, administrative costs, enrollment volume, growth opportunities in other markets) that matter rather than simply the level of rates.

In vitro modulation of the firing rate of dopamine neurons in the rat substantia nigra pars compacta and the ventral tegmental area by antipsychotic drugs.

An in vitro experimental midbrain slice preparation is described which allows simultaneous extracellular recordings of the (spontaneous) electrical activity of dopamine neurons in the rat substantia nigra (SN) and the ventral tegmental area (VTA). Under identical in vitro circumstances the mean firing frequency of the SN dopamine neurons was higher than that of the VTA dopamine neurons (2.1 vs. 1.4Hz). With this slice preparation, modulation of the electrical activity of SN and VTA dopamine neurons by (new) drugs can be quickly determined. Experiments with the selective D2 receptor agonist quinpirole and the selective D2 receptor antagonist (-)-sulpiride indicated that dopamine neurons in the SN and VTA hardly differ in their pharmacological properties for the D2-like (auto)receptor. (-)-Sulpiride and to a lesser extent risperidone induced a small increase in firing rate in SN and VTA neurons, which was reversible upon wash-out. Olanzapine-induced increase in firing rate was persistent in SN and VTA neurons, whereas the clozapine-induced increase in firing rate was only completely recovered upon wash-out in SN neurons. The difference in firing rates of SN and VTA dopamine neurons could have consequences for the effectiveness of dopaminergic drugs acting at the D2-like dopamine (auto)receptor on these neurons.

Impacts of Medicaid managed care on children.

OBJECTIVE: To assess the impact of switching from a fee-for-service (FFS) delivery system to managed care on access to, use of, and satisfaction with health care for children. DATA SOURCES/STUDY SETTING: A 1998 survey of Medicaid recipients in rural Minnesota. STUDY DESIGN: Using a quasi-experimental framework, we compare the experiences of children on Medicaid living in counties that had switched to managed care with those of children living in counties operating under FFS Medicaid. We address the impact of Medicaid managed care (MMC) on access to, use of, and satisfaction with care. DATA COLLECTION METHODS: A stratified random sample of children on Medicaid was drawn based on Medicaid enrollment files. Telephone interviews were conducted with the child's parent or guardian between March and June 1998. An overall response rate of 70 percent was achieved, yielding a sample of 1,106 children (814 in MMC and 792 in Medicaid FFS). PRINCIPAL FINDINGS: We find very few significant differences in access to, use of, or satisfaction with health care services for children under MMC relative to FFS. MMC did not change the patterns of health care service use or the location at which care is delivered, two major goals of MMC initiatives. CONCLUSIONS: Our results suggest that the Medicaid program's shift from FFS to managed care had little impact on the pattern of children's health care use, the location at which they obtained care, or the satisfaction with the care they received.

Antipsychotic drugs classified by their effects on the release of dopamine and noradrenaline in the prefrontal cortex and striatum.

Dose-effect curves were established for the effects of the antipsychotic drugs haloperidol, clozapine, olanzapine, risperidone and ziprasidone on extracellular levels of dopamine and noradrenaline in the medial prefrontal cortex, and of dopamine in the striatum. Haloperidol was more effective in stimulating the release of dopamine in the striatum, whereas clozapine was much more effective in the medial prefrontal cortex. The efficacy of risperidone, olanzapine and ziprasidone did not differ for the two brain areas. The benzamides sulpiride and raclopride increased dopamine release in the striatum but did not affect the release of dopamine and noradrenaline in the medial prefrontal cortex. In the presence of dopamine/noradrenaline reuptake inhibitors, the benzamides strongly increased the release of dopamine-but not of noradrenaline-in the medial prefrontal cortex. The 5-HT(2) receptor antagonist R-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol (MDL100,907) (800 nmol/kg) and the dopamine D(2) receptor antagonist raclopride (2 micromol/kg) displayed a clear synergism in increasing the release of dopamine in the medial prefrontal cortex. No such synergism was seen in the case of noradrenaline. Co-administration of the 5-HT(2) receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine HCl (DOI) (850 nmol/kg) with clozapine (10 micromol/kg) or haloperidol (800 nmol/kg) blocked the increase in dopamine as well as noradrenaline in the medial prefrontal cortex. It is concluded that typical and non-benzamide atypical antipsychotics increase extracellular dopamine in the medial prefrontal cortex via a synergistic interaction by blocking 5-HT(2) as well as dopamine D(2) receptors. The increase in extracellular noradrenaline in the medial prefrontal cortex that was observed after administration of antipsychotics is explained by inhibition of 5-HT(2) receptors and not dopamine D(2) receptors. Finally, the significance of the classification of antipsychotic drugs based on their selective action on the release of dopamine and noradrenaline in the medial prefrontal cortex is discussed. In particular, the position of the benzamides is discussed.

Access and use by children on Medicaid: does state matter?

Although Medicaid is a central component of health care for children, the program is not uniform across the states. Using state and nationally representative data from the 1997 National Survey of America's Families (NSAF), a survey of the economic, health and social characteristics of children, nonaged adults and their families, we examine differences in access and use by children on Medicaid across 13 states, and compare those differences to national estimates. We find significant differences in access and use across the states for children on Medicaid. The characteristics of the children and their local health care environment explain some, but not all, of the state differences in access and use.

How do they manage? Disabled elderly persons in the community who are not receiving Medicaid long-term care services.

OBJECTIVE: To expand our understanding of how low-income functionally impaired elderly persons are able to remain in the community. DATA SOURCES AND STUDY SETTING: In-person and telephone interviews with 25 elderly individuals who applied for but did not enroll in Connecticut's Home Care Program for Elders (CHCPE). All met the state's nursing home level-of-care criteria. STUDY, DESIGN: In-depth discussions with a small, purposefully selected sample of functionally impaired elderly persons in the community. PRINCIPLE FINDINGS: Many sample members with very high levels of impairment and multiple chronic health conditions remained in the community without CHCPE services because of Medicare home health services combined with extensive levels of informal care. Some sample members, particularly those with more limited informal care networks, did not receive the level of care that they needed. Virtually all were at high risk for medical complications, hospitalizations for acute illnesses, falls, and further loss of functioning. Further, in many cases, informal care networks were overextended, stressed and vulnerable to break down. All but a few of those we interviewed were not receiving services through the waiver program for financial reasons. Most met Medicaid's income criteria but had assets that exceeded Medicaid's $2,000 limit. Several were not participating due to concerns about estate recovery. CONCLUSIONS: Additional formal help is needed to avoid eventual nursing home placement for many sample members. This could be achieved by expanding the availability of case management services and/or relaxing program financial requirements. Further, efforts to reduce Medicare home health expenditures must recognize the heightened vulnerability of many beneficiaries for potentially costly adverse outcomes.

Interactions between Medicare and Medicaid home care in Connecticut: responses to the 1997 BBA.

Although overlaps exist in the provision of home care services financed by Medicare and Medicaid, interactions of the two funding sources at the beneficiary and home care provider levels have not been widely studied. The recent dramatic declines in Medicare home health spending present a rare opportunity to examine how changes in Medicare spending can affect the provision of Medicaid home care services. We conducted a study of Medicare-Medicaid dynamics in the state of Connecticut to shed light on the inter-relationships of the two funding sources.

Glucose depletion rapidly inhibits translation initiation in yeast.

Glucose performs key functions as a signaling molecule in the yeast Saccharomyces cerevisiae. Glucose depletion is known to regulate gene expression via pathways that lead to derepression of genes at the transcriptional level. In this study, we have investigated the effect of glucose depletion on protein synthesis. We discovered that glucose withdrawal from the growth medium led to a rapid inhibition of protein synthesis and that this effect was readily reversed upon readdition of glucose. Neither the inhibition nor the reactivation of translation required new transcription. This inhibition also did not require activation of the amino acid starvation pathway or inactivation of the TOR kinase pathway. However, mutants in the glucose repression (reg1, glc7, hxk2, and ssn6), hexose transporter induction (snf3 rgt2), and cAMP-dependent protein kinase (tpk1(w) and tpk2(w)) pathways were resistant to the inhibitory effects of glucose withdrawal on translation. These findings highlight the intimate connection between the nutrient status of the cell and its translational capacity. They also help to define a new area of posttranscriptional regulation in yeast.

Neurotensin attenuates the quinpirole-induced inhibition of the firing rate of dopamine neurons in the rat substantia nigra pars compacta and the ventral tegmental area.

In the present study we describe the excitatory effects of the bioactive peptide neurotensin on the electrical activity of dopamine neurons (simultaneously recorded) in the substantia nigra pars compacta and the ventral tegmental area. The neurotensin fragment (8-13) induced comparable increases in firing rate of the substantia nigra and ventral tegmental area dopamine neurons (EC50 values 30 and 45 nM, respectively). The neurotensin receptor antagonist SR142948A antagonized the excitatory effects of neurotensin fragment (8-13) (pA2 values 8.4 and 8.2, respectively). Furthermore, it was found that a low concentration of neurotensin fragment (8-13) (1 nM) attenuated the inhibition of the firing rate by the selective dopamine D2 receptor agonist quinpirole in both neuron types (e.g., the effect of 0.01 microM quinpirole was reduced by approximately 60% in the presence of 1 nM neurotensin fragment [8-13]). Antagonism of this neurotensin fragment (8-13) effect by SR142948A confirms that neurotensin receptors can reduce the effect of dopamine D2 receptors at the single-cell level. These results are discussed in the light of possible roles for neurotensin in neurological disorders such as Parkinson's disease and schizophrenia.

Effects of medicaid managed care on adults.

BACKGROUND: Despite the rapid growth in Medicaid managed care (MMC) during the 1990s, only limited research exists on how such care affects beneficiaries. OBJECTIVE: The objective of this study was to assess how switching from a fee-for-service (FFS) delivery system to managed care affects Medicaid beneficiaries' access to, use of, quality of, and satisfaction with health care services. METHODS: Using a quasi-experimental design framework, we compared the experiences of 540 Minnesota Medicaid recipients living in counties that had switched to managed care with those of 528 recipients living in counties operating under FFS. The data for the analysis came from a 1998 survey of Minnesota Medicaid clients. Data were analyzed by logit regression. RESULTS: We find limited effects of MMC on access to, use of, quality of, and satisfaction with health care. Among others, we found no significant differences between the share of managed care and FFS enrollees (78.5% versus 76%) who had a health care visit during the last year. We also found no evidence of a significant reduction in the proportion of managed care and FFS enrollees (17.6% versus 17%) who had had a hospital stay during the past year. The results did show some negative effects of MMC on satisfaction with care, the most consistent being that managed care enrollees are somewhat less satisfied with their health care than their FFS counterparts. CONCLUSIONS: Our results suggest that a shift from FFS to MMC did not fundamentally change the patterns of health care service use, the location at which care was delivered, or quality.

Antipsychotic drugs induce similar effects on the release of dopamine and noradrenaline in the medial prefrontal cortex of the rat brain.

In the present study we have compared the effects of the classical antipsychotic drug haloperidol and four different atypical antipsychotics (clozapine, risperidone, olanzapine, ziprasidone) on extracellular levels of dopamine and noradrenaline in the medial prefrontal cortex (MPFC) of conscious rats. Haloperidol (10, 100 and 800 nmol/kg), clozapine (0.3, 1, 10 and 30 micromol/kg), risperidone (100, 500 and 5000 nmol/kg), olanzapine (10, 100 and 500 nmol/kg) and ziprasidone (10, 100 and 1000 nmol/kg) were administered subcutaneously to rats. All compounds induced increases in dialysate levels of dopamine and noradrenaline in the medial prefrontal cortex. The increases induced by the four antipsychotic agents in extracellular levels of dopamine and noradrenaline displayed a striking co-variation both in dose and time. A similar co-variation was seen in the decrease of dopamine and noradrenaline, after administration of a low dose (30 nmol/kg, s.c.) of the dopamine D2/3 receptor agonist (+)-7-hydroxy-2-(N,N-di-n-propylamino) tetralin ((+)-7-OH-DPAT). It is concluded that there is a close coupling between the release of dopamine and noradrenaline in the medial prefrontal cortex. The mechanism of action of this interaction, that might be of importance for a better understanding of the mechanism of action of antipsychotic drugs, is discussed.

Preferential blockade of cholecystokinin-8S-induced increases in aspartate and glutamate levels by the CCK(B) receptor antagonist, L-365,260, in rat brain.

In the present studies, the ability of a locally delivered cholecystokinin (CCK) receptor agonist and systemically delivered antagonists to modulate extracellular levels of aspartate and glutamate in the frontal cortex of anaesthetised rats and frontal cortex and caudate-putamen of freely moving rats was investigated using an in vivo microdialysis technique. In the anaesthetised rats, local application of sulphated CCK octapeptide (CCK-8S, 10 microM) into the frontal cortex enhanced extracellular aspartate levels to a maximum of 265+/-16% of the basal levels, whereas glutamate levels were increased to a maximum of 168+/-7% of the basal levels. Given 40 min prior to the cortical perfusion of 10 microM of CCK-8S, the CCK(B) receptor antagonist, L-365,260 (20 mg/kg, s.c.), limited the rise in cortical aspartate by over half to 170+/-10% of the basal levels. However, this same dose of L-365,260 still allowed CCK-8S to increase glutamate by 44+/-15% above the basal levels. Whereas the enhanced glutamate levels were totally unaffected by systemic administration of the CCK(A) receptor antagonist, L-364,718 (20 mg/kg, -40 min, s.c.), this treatment was able to limit the elevation in aspartate to 220+/-4% of the basal levels. In the freely moving rats, local perfusion of CCK-8S (10 microM) increased aspartate and glutamate levels to maxima of 275+/-12% and 225+/-14% of the basal levels, respectively, in the frontal cortex. In the caudate-putamen, aspartate and glutamate levels were also elevated by CCK-8S (10 microM) to 248+/-15% and 185+/-12% of the basal levels, respectively. The respective increase in aspartate and glutamate induced by CCK-8S (10 microM) were limited to 140+/-10% and 124+/-6% (frontal cortex), of the basal levels, and 162+/-15% and 143+/-8% (caudate-putamen), by 40 min pretreatment with L-365,260 (20 mg/kg, s.c.). In conclusion, CCK-8S was able to enhance both aspartate and glutamate overflow in the frontal cortex of anaesthetised rats, and frontal cortex and caudate-putamen of freely moving rats. These increases were preferentially offset by the selective CCK(B) receptor antagonist, L-365,260, since no influence could be discerned using the selective CCK(A) receptor antagonist, L-364,718.

Urban health care in transition: challenges facing Los Angeles County.

The authors examine the Medicaid Section 1115 Demonstration Project currently underway in Los Angeles County. The waiver was designed as part of a response to a financial crisis the Los Angeles County Department of Health Services (LACDHS) faced in 1995. It provides financial relief to give the county time to restructure its system for serving the medically indigent population. Los Angeles County's goal is to reduce its traditional emphasis on emergency room and hospital care by building an integrated system of community-based primary, specialty, and public health care. This case study describes activities completed through the spring of 1997, approximately 1 year after the waiver was approved.

The effects of central myorelaxants on synaptically-evoked primary afferent depolarization in the immature rat spinal cord in vitro.

1. In the immature rat in vitro hemisected spinal cord preparation the dorsal root-evoked depolarizing potential recorded from an adjacent dorsal root DR-DRP had a mean peak amplitude (+/- s.e.mean, n = 27) of 2.9 +/- 0.2 mV and a mean latency to peak amplitude of 106 +/- 3 ms. The DR-DRP amplitude was maximal with a stimulus intensity of four times the threshold intensity required to activate the lowest threshold fibres. The peak amplitude and/or integral over a time-source of 0.5 s were used to assess the effects of applied drugs. 2. The DR-DRP was abolished by baclofen (mean IC50 190 +/- 46 nM, n = 7). The depressant effect of baclofen was reversed by CGP35348 (1 mM). The mean apparent Kd value calculated from dose-ratios was 16.7 +/- 6.4 microM (n = 3). 3. At a maximally effective concentration, tizanidine (1 microM) produced at the most only a 14% depression of the DR-DRP (n = 4). Clonidine (0.3 microM) had an effect similar to that of tizanidine. These depressant effects were reversed by idazoxan (1 microM). 4. The DR-DRP was potentiated by diazepam in a flumazenil (1 microM)-reversible manner. A maximal potentiation of 23.2 +/- 2.7% (n = 5) was produced by 1 microM diazepam. 5. Diazepam (1 microM) induced a mean bicuculline- (10 microM, n = 2) and flumazenil- (1 microM, n = 8) sensitive depolarization in the dorsal root of 0.25 +/- 0.03 mV (n = 8). However, diazepam failed to depolarize dorsal roots (n = 3) which had been excised from the spinal cord. 6. Comparison of the above effects with previously reported depressant effects of these drugs on the synaptic output from ventral roots suggests that actions on presynaptic inhibition, as reflected in the DR-DRP, are of subsidiary importance in explaining the muscle relaxant actions of tizanidine or diazepam.